Some Success

Here is one example of the work I have been doing over the past few weeks:

This past week I got the result from my western Blot. After the difficulties with the first bisulfite conversion, it was a nice change to see some expected results. In this western blot we used MCF7 cells and probed using primary antibodies for Tubulin, Ecad, B4, and Actin. The secondary antibodies were Rabbit, Rat, and Mouse. These antibodies bind to the sites of the proteins. After developing the results one can see/compare the amount of proteins in each phase of the cell. The two groups of cells we looked at were Hypoxia and Normoxia. Normoxia is the characterization of cells that have grown in normal oxygen levels. Hypoxia is the characterization of cells that have grown in an atmosphere of 0.5% oxygen. This is achieved by using a machine that keeps the conditions at 0.5% oxygen. In our results, we saw the Ecad decreased from normoxia to Hypoxia, and Beta4 also decreased from normoxia to Hypoxia.

To give an idea of what a western blot entails this is the protocol I had to follow:

1. Make the Acrylamide gel
-two separate gels-stacking gel and Resolving gel-the stacking gel is where you load the samples and the resolving gel is where the samples pass through when an electric current is added.

2. Prepare the samples- Acrylamide gels are for proteins. After extracting a lysate from the cells, you measure the concentration using a bradford machine. Using those concentrations you prepare each sample with equal volume of proteins. If the concentrations differ, the volume will differ. In this case you add Ripa buffer to the lesser volume to make them equal.

3. Add 6x buffer to each sample

4. Load/run the gel. Load gel with Markers as well.

5. With an 8% gel it usually takes about 20-25 Minutes to run.


After the gel runs you transfer the samples on the gel to a membrane using a transfer aparatus. You run this in a cold room at 350 Amps for 90 Minutes.

This is only the first half of a long process. Later in the experiment you have to probe for the proteins and develop the results in a dark room.

I have had success with the western blots and am doing another one today.



Overall the past few days have been really busy. I have been running a lot of experiments, and am learning a tremendous amount. Next week, I will be giving my presentation to the lab on the data I have been getting.